Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The overall aim of this procedure is to create compositionally representative, multi-species dental plaque biofilms. In parallel for analysis, this is achieved by first creating a representative media and inoculum. The inoculum is then introduced to the microfluidic chip after an overnight incubation, a biofilm forms inside the micro channel.

Finally, the biofilm is washed and stained for in situ two confocal laser scanning microscopy or epi fluorescence microscopy. The main advantage to this technique over existing methods like flow cells is that it's a high throughput system that allows for multiple experiments to be done in parallel while using less materials and not requiring artificial lab media. To begin collect saliva samples from a cohort of five or more volunteers in individual 50 milliliter plastic tubes.

Pull the saliva samples into a single plastic beaker placed on ice. Avoid using glass beakers in this setup as saliva biopolymers tend to adhere to glass surfaces. Next, add di thio threes hole to the sample until its final concentration is 2.5 millimolar.

Stir the mixture for 10 minutes on ice. Pull all contents into several plastic tubes. Perform a high speed spin down for 30 minutes in a four degrees Celsius chilled centrifuge to separate particulates from the sample.

Transfer the saliva sate to a fresh, sterile container and discard the pellet fraction to prepare a bacteria free growth medium from the sample. First, dilute the day debris free sample with three volumes of deionized water. Then using a 0.22 micro low protein binding membrane filter.

Sterilize the saliva while keeping the post filtered fraction chilled on ice. Eloquent the sterilized solution and store at minus 80 degrees Celsius until needed. Collect saliva samples from a cohort of five or more volunteers in sterile 50 milliliter plastic tubes.

Pull the saliva samples into a room temperature pre sterilized plastic beaker, or 50 milliliter tube. Add sterilized glycerol until a 25%glycerol. 75%saliva mixture is reached.

Unlike the growth medium protocol where contaminants were removed with a filtration step, sterile technique is important here to prevent contamination from non-oral specific bacteria or fungi. Mix the saliva glycerol solution. Well aliquot the saliva glycerol mixture and store samples at minus 80 degrees Celsius until they are needed to begin.

Purchase or fabricate a microfluidic chip with microchannel similar to those shown here prior to inoculating samples into the microchip for both the bacteria negative growth medium and bacteria positive inoculum on the bench at room temperature. Vortex each tube for five seconds to mix before proceeding for the main experiment, start by adding 100 microliters of the growth medium at the microchips outlet reservoir. Using a computer control pump commence medium sheer flow through the microchannel for two minutes.

At room temperature, visually inspect each channel to ensure proper fluid filling. Incubate the microchip at room temperature for 20 minutes. This will coat the microchannel sidewalls with a biopolymer scaffold to which the biofilm will anchor onto during growth.

Then manually aspirate or remaining media from the outlet reservoir and transfer this fraction, which will now act as a hydrodynamic balancing load to the inlet reservoir. After biopolymer scaffold deposition, add 100 microliters of the bacteria positive inoculum to the outlet reservoir. Place the microchip onto a 37 degree Celsius hot plate and commence medium shear reverse flow.

Traveling from the outlet to the inlet Reservoir for exactly six seconds. Then turn the pump off and incubate the chip under static conditions at 37 degrees Celsius for 40 minutes to facilitate attachment of bacteria or fungi to the microchannel side walls. Next, remove and discard all inoculum from the outlet reservoir with a pipette and refill the same reservoir by adding one milliliter of bacteria free growth medium.

Incubate the microchip at 37 degrees Celsius for approximately 20 hours under low sheer flow to promote intra channel biofilm growth upon overnight growth. Pipette out all solutions from both inlet and outlet reservoirs. Apply 100 microliters of PBS at the Inlet Reservoir and wash the micro channel for 20 minutes.

Under low sheer forward flow from the inlet to the outlet, the biofilm is now ready for live dead staining. Just prior to biofilm staining, prepare 100 microliters of the viability staining mix as found in the text protocol for every channel to be stained. Protect this solution from light exposure until it is needed to stain the biofilm aspirate all remaining liquid from the inlet and refill the same reservoir with 100 microliters of the viability staining mix.

Then push the staining solution through the microchannel for 45 minutes. Under low share flow at room temperature, be sure to protect the chip from light exposure. Aspirate all remaining liquid from the inlet reservoir and refill with 100 microliters of PBS.

Wash the microchannel for 20 minutes Under low share flow at room temperature to remove excess unbound dyes. The stained biofilm is now ready for fluorescent microscopy to assay for the biofilms three dimensional architecture. Digital reconstructions using horizontal scans with a confocal microscope can be used to view the overall film structure from any oblique angle.

Moreover, live dead staining analysis can be applied to the same data dataset to study the spatial dependence of cell viability inside the biofilm as a function of different chemical treatments. Once the biofilm has been imaged, cells from unstained channels can be extracted. Using distilled water operating at high shear flow, the biofilm flora can then be identified from the genomic DNA by 4 5 4 pyro sequencing.

Following this procedure. Other methods like adding different species or compounds can be performed in order to answer different questions such as what happens to the multi-species biofilms under various conditions.